Figure 7.

ATR phosphorylates SMARCAL1 on S652 after SMARCAL1 binds to DNA at stalled forks. (A,B) HEK293T cells were transfected with small amounts of GFP-SMARCAL1 wild-type (WT) and mutant expression vectors to minimize overexpression. ΔH1 and ΔH2 are deletions of the HARP1 and HARP2 domains, respectively, while H1-WF and H2-WF are point mutants in each HARP domain (Betous et al. 2012). Cells were treated with 2 mM HU for 16 h where indicated prior to lysis, separation by SDS-PAGE, and immunoblotting with SMARCAL1 antibody. (C) SMARCAL1 was purified from untreated (unt.) or HU-treated (2 mM, 16 h) HEK293T cells and was used to measure ATPase activity in the presence of 5 nM forked DNA substrate. Error bars are standard deviation from three experiments (P = 0.0007, two-tailed unpaired t-test). The inset is an immunoblot showing equal amounts of protein used in each condition. The purified protein was treated with λ phosphatase before immunoblotting to eliminate the gel mobility shift and allow more accurate quantitation of protein concentration. (D) ClustalW was used to align SMARCAL1 from the indicated organisms. The position of the phosphorylated SQ residues S173, S652, and S919 relative to protein domains is depicted. (E,F) HEK293T cells were transfected with Flag-SMARCAL1 and treated with HU for the indicated times. Kinase inhibitors were added as indicated. Flag-SMARCAL1 was immunoprecipitated from cell lysates, separated by SDS-PAGE, and immunoblotted with either total SMARCAL1 antibody or pS652-specific antibody. (*) Nonspecific bands. Images were captured and quantitated relative to total SMARCAL1 using an Odyssey imaging system. (G) Purified ATR–ATRIP (ATR-interacting protein) complex phosphorylates a GST-S652 peptide in vitro. ATRi was added where indicated to ensure specificity of the kinase in the reaction. Shown are images of a Coomassie-stained gel to visualize the amount of ATR and GST protein in the reactions or an autoradiogram (³²P) of the gel to visualize phosphorylation.